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Solubilization and humanization of paraoxonase-1.

Sarkar M, Harsch CK, Matic GT, Hoffman K, Norris JR, Otto TC, Lenz DE, Cerasoli DM, Magliery TJ - J Lipids (2012)

Bottom Line: Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1.All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most.Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

No MeSH data available.


Related in: MedlinePlus

PON1 solubilizing mutations. (a) The surface of G2E6 is shown, with hydrophobic amino acids (VGMCILYFW) shown in red. Residues 1–15 are not resolved in the X-ray crystal structure, but the ΔN-huPON1 variant removed residues 4–17. (b) The positions modified in the ΔHDL-huPON1 variant are shown in spheres. These residues compose much of the hydrophobic surface patch near the N-terminus evident in (a). The Ca2+ ions are shown as pink spheres, and a phosphate bound in the presumed active site is shown in orange sticks. (c) The 59 positions that differ between huPON1 and G2E6 are shown; the positions that were modified in g2e6p-huPON1 are spheres, and the other 43 positions are sticks. Position 166, which was modified because of its proximity of 192, is noted. Rendered from PDB ID: 1V04 with PyMOL.
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fig3: PON1 solubilizing mutations. (a) The surface of G2E6 is shown, with hydrophobic amino acids (VGMCILYFW) shown in red. Residues 1–15 are not resolved in the X-ray crystal structure, but the ΔN-huPON1 variant removed residues 4–17. (b) The positions modified in the ΔHDL-huPON1 variant are shown in spheres. These residues compose much of the hydrophobic surface patch near the N-terminus evident in (a). The Ca2+ ions are shown as pink spheres, and a phosphate bound in the presumed active site is shown in orange sticks. (c) The 59 positions that differ between huPON1 and G2E6 are shown; the positions that were modified in g2e6p-huPON1 are spheres, and the other 43 positions are sticks. Position 166, which was modified because of its proximity of 192, is noted. Rendered from PDB ID: 1V04 with PyMOL.

Mentions: To generate ΔHDL-huPON1 (Figures 1 and 3), twelve Glu, Gln, or Lys mutations were made at hydrophobic residues in the putative HDL binding site (Y24E, Y185E, F186Q, L187K, Y190K, L191Q, W194K, L198E, L200Q, W202K, M289Q, F293E) in the huPON1 construct. All of these mutations were introduced at once by overlap PCR. Three pairs of oligonucleotides were used to amplify three fragments of the huPON1 gene with all 12 mutations. The three fragments were then subjected to assembly and amplification similar to the final steps of DNA shuffling using two terminal primers, 5′-ATAGATATAC ATATGGCGAA GCTGATTGCA CTCACGCTCT TGGGGATGGG ACTGGC ACTC TTCAGGAACC ACC-3′ and 5′-CTCACCGCCG GTACCGAGTT CGCAGTAAAG AGCTTTG-3′. These primers coded for NdeI and KpnI sites. The amplified ΔHDL-huPON1 gene was cloned into pET11-huPON1-TEV-frGFP vector, replacing huPON1 between NdeI and KpnI, and the construct was confirmed by DNA sequencing.


Solubilization and humanization of paraoxonase-1.

Sarkar M, Harsch CK, Matic GT, Hoffman K, Norris JR, Otto TC, Lenz DE, Cerasoli DM, Magliery TJ - J Lipids (2012)

PON1 solubilizing mutations. (a) The surface of G2E6 is shown, with hydrophobic amino acids (VGMCILYFW) shown in red. Residues 1–15 are not resolved in the X-ray crystal structure, but the ΔN-huPON1 variant removed residues 4–17. (b) The positions modified in the ΔHDL-huPON1 variant are shown in spheres. These residues compose much of the hydrophobic surface patch near the N-terminus evident in (a). The Ca2+ ions are shown as pink spheres, and a phosphate bound in the presumed active site is shown in orange sticks. (c) The 59 positions that differ between huPON1 and G2E6 are shown; the positions that were modified in g2e6p-huPON1 are spheres, and the other 43 positions are sticks. Position 166, which was modified because of its proximity of 192, is noted. Rendered from PDB ID: 1V04 with PyMOL.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376767&req=5

fig3: PON1 solubilizing mutations. (a) The surface of G2E6 is shown, with hydrophobic amino acids (VGMCILYFW) shown in red. Residues 1–15 are not resolved in the X-ray crystal structure, but the ΔN-huPON1 variant removed residues 4–17. (b) The positions modified in the ΔHDL-huPON1 variant are shown in spheres. These residues compose much of the hydrophobic surface patch near the N-terminus evident in (a). The Ca2+ ions are shown as pink spheres, and a phosphate bound in the presumed active site is shown in orange sticks. (c) The 59 positions that differ between huPON1 and G2E6 are shown; the positions that were modified in g2e6p-huPON1 are spheres, and the other 43 positions are sticks. Position 166, which was modified because of its proximity of 192, is noted. Rendered from PDB ID: 1V04 with PyMOL.
Mentions: To generate ΔHDL-huPON1 (Figures 1 and 3), twelve Glu, Gln, or Lys mutations were made at hydrophobic residues in the putative HDL binding site (Y24E, Y185E, F186Q, L187K, Y190K, L191Q, W194K, L198E, L200Q, W202K, M289Q, F293E) in the huPON1 construct. All of these mutations were introduced at once by overlap PCR. Three pairs of oligonucleotides were used to amplify three fragments of the huPON1 gene with all 12 mutations. The three fragments were then subjected to assembly and amplification similar to the final steps of DNA shuffling using two terminal primers, 5′-ATAGATATAC ATATGGCGAA GCTGATTGCA CTCACGCTCT TGGGGATGGG ACTGGC ACTC TTCAGGAACC ACC-3′ and 5′-CTCACCGCCG GTACCGAGTT CGCAGTAAAG AGCTTTG-3′. These primers coded for NdeI and KpnI sites. The amplified ΔHDL-huPON1 gene was cloned into pET11-huPON1-TEV-frGFP vector, replacing huPON1 between NdeI and KpnI, and the construct was confirmed by DNA sequencing.

Bottom Line: Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1.All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most.Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

No MeSH data available.


Related in: MedlinePlus